Generation of high-quality circular nucleic acids is desirable for nucleic acid based therapeutic applications, research involving transformation or transduction of cell lines, and the like. For example, deoxyribonucleic acid (DNA)-based therapeutics in gene therapy, gene transfer, and DNA vaccination demand large-scale generation of DNA having stringent quality criteria in terms of high purity, potency, efficacy, and safety.
Linear DNA molecules are rapidly degraded by nucleases, limiting their use for DNA-based therapeutic applications such as vaccination. Most of the currently available DNA therapeutic applications therefore use circular nucleic acids or plasmids. Supercoiled DNA plasmids are particularly beneficial for such applications since they are not easily degraded by the nucleases. These circular nucleic acids or plasmids are usually grown in bacterial cell culture, and their purification from the bacterial cells often employ hazardous or toxic reagents. Such plasmid preparation procedures therefore carry a potential risk of contamination in terms of toxic reagents, transposes and other episomal DNA, residual host cell nucleic acids, residual host cell proteins, endotoxins, and the like. To meet the quality criteria required for nucleic acid-based therapeutics, extensive purification techniques are often required, which are laborious, time-consuming, and expensive.
Cell-free nucleic acid amplification techniques provide a viable alternative for generating high quality nucleic acids that are devoid of any bacterial contamination. Such in vitro nucleic acid amplification techniques also have significant advantages in terms of cost savings, streamlined production, and simplified purification. However, some in vitro nucleic acid amplification methods, such as polymerase chain reaction (PCR), require quick thermal cycling, and so are often not amenable for large-scale generation of high-quality nucleic acids. Moreover, PCR products, being linear DNA sequences, are rapidly degraded in a host by the action of nucleases. In contrast, isothermal nucleic acid amplification techniques such as rolling circle amplification (RCA) or strand displacement amplification (SDA) may be employed to generate high-quality nucleic acids with less effort and expense. RCA typically produces concatamers comprising linear tandem repeat units of input circular nucleic acid template sequence. These tandem repeat sequences are useful for routine molecular biology experiments such as cloning and sequencing. However, they are seldom used in nucleic acid-based therapeutics because the transformation or transfection efficiencies of these concatamers are often lower. Currently known methods used to convert concatamers to circular nucleic acids (mini-circles) require multiple steps involving multiple enzymatic reactions. For example, concatamers may be first cut into small fragments using restriction enzymes, and then re-ligated using ligases to generate circular nucleic acids. There exists a need for efficient methods for large-scale production of high-quality circular nucleic acids that are optimally free of any bacterial sequences and contaminants.